1. Field of the Invention
The present invention relates to a microbiological process for eliminating a nitroaromatic compound present in a solution or in a soil, and to a microorganism strain capable of mineralising said nitroaromatic compound.
The method according to the invention is for example very useful for treating a solution, such as an industrial effluent, or a soil, polluted with a nitroaromatic compound, for example with dinitrotoluene (DNT) or trinitrotoluene (TNT).
2. State of the Related Art
Different nitroaromatic compound degradation processes have been studied in the prior art. These processes are essentially either biological or chemical.
For example, in 1998, KALAFUT et al. studied the transformation of TNT by three aerobic bacteria: Pseudomonas aeruginosa, Bacillus sp. and Staphylococcus sp. It demonstrated that these three strains transformed TNT but could not use it as the only source of carbon or nitrogen. The study is reported in the document KALAFUT, T et al., xe2x80x9cBiotransformation patterns of 2,4,6-trinitrotoluene by aerobic bacteriaxe2x80x9d, Cur. Microbiol., 1998, 36, 45-54.
In 1998, BOOPATHY et al. developed a laboratory-scale aerobic/anoxic bioreactor to decontaminate a soil contaminated with TNT. This method may be carried out in a batch or semi-continuous system. Batch treatment enables TNT transformation, but some metabolites accumulate. However, in semi-continuous mode (regular change of 10% of soil), TNT (8 g/kg of soil) is completely broken down. The balance, after a 14-day incubation period, indicates that only 23% of the TNT was mineralised, 27% assimilated and 8% was adsorbed on the soil. The remaining percentage corresponds to the formation of metabolites such as amino-dinitrotoluenes (4%), 2,4-diamino-6-nitrotoluene (3%) and 2,3-butanediol produced by the splitting of the cycle (30%). Successive additions of soil once, twice or three times a week do not affect the TNT degradation rate. In addition, this reactor makes it possible to maintain the bacterial population, with 0.3% molasses being the only co-substrate required. Using the same method, it is possible to eliminate other soil contaminants, such as hexahydro-1,3,5-trinitro-1,3,4-triazine (RDX), octahydro-1,3,5,7-tetranitro-1,3,5,7-tetrazocine (HMX), trinitrotoluene and 2,4-dinitrotoluene. These results are reported in BOOPATHY, R. et al. xe2x80x9cA laboratory study of the bioremediation of 2,4,6-trinitrotoluene-contaminated soil using aerobic/anoxic soil slurry reactorxe2x80x9d, Water Environment Research, 1998.
In 1998, VORBECK et al. studied the microbiological reduction of TNT nitro groups and the microbiological hydrogenation of the aromatic cycle. Due to electron deficiency caused by the presence of electron-attracting nitro groups, the first microbial transformation of TNT is a nitro-reduction. The hydrogenation of the aromatic cycle described for picric acid remains a minority. Two bacteria were isolated from an aerobic medium, enriched with TNT as the only source of nitrogen: the strain TNT-8 (gramxe2x88x92) and the strain TNT-32 (gram+). They catalyse the nitro-reduction of TNT. However, the strains Rhodoccus erythropolis HL PM-1 (growing on picric acid) and Mycobacterium sp. HL 4-NT-1 (growing on 4-nitrotoluene) with enzyme systems which catalyse the hydrogenation of TNT and, consequently, the addition of a hydride ion on the aromatic cycle. The TNT Meisenheimer hydride complex (H-TNT) formed is then converted into a yellow non-degradable metabolite, identified by NMR. In this case, no reductive denitration of the TNT was observed. The mineralisation of TNT was not studied. These results are reported in VORBECK, C. et al., xe2x80x9cInitial Reductive reactions in aerobic microbial metabolism of 2,4,6-trinitrotoluenexe2x80x9d, Appl. Environ. Microbiol., 1998, 64(1), 246-252.
However, none of the micro-organisms described in these documents enable the total or almost total mineralisation of TNT.
Chemical degradation processes, for their part, involve numerous disadvantages related to the use of chemical reagents. These disadvantages particularly include the cost of the chemical reagents and the pollution generated by these reagents in treated solutions and soils.
The specific aim of the present invention is to provide a method enabling the total or almost total elimination of a nitroaromatic compound in a solution or in a soil, by means of the mineralisation of said compound.
In this way, the method according to the present invention is a method which may be used to treat a solution or a soil containing one or more undesirable (due to pollution) nitroaromatic compounds.
The method according to the invention is a microbiological method for eliminating at least one nitroaromatic compound present in a solution or in a soil, said method being characterised in that it comprises contact of said solution or said soil with a biomass of a Penicillium strain under suitable conditions for mineralisation of the nitroaromatic compound by the Penicillium strain.
The terms xe2x80x9cnitroaromatic compoundxe2x80x9d or xe2x80x9cnitroaromatic compound solutionxe2x80x9d below refer to xe2x80x9cthe nitroaromatic compound(s)xe2x80x9d and xe2x80x9csolution of the nitroaromatic compound(s)xe2x80x9d, respectively.
According to the invention, the nitroaromatic compound solution may be, for example, a laboratory solution, an industrial effluent or surface water containing one or more nitroaromatic compound(s) and wherein, preferentially, the Penicillium strain can metabolise the nitroaromatic compound. Advantageously, according to the method of the invention, this solution is an aqueous solution.
According to the invention, the, at least one, nitroaromatic compound may be a compound comprising at least one aromatic cycle comprising at least one nitro group and, if applicable, at least one function selected in the group comprising xe2x80x94OH, xe2x80x94COOH, a halogen, xe2x80x94NH2, a cyclic or linear ose, a linear or ramified alkyl comprising 1 to 12 carbon atoms or an aryl, not substituted or substituted, by at least one function selected in the group comprising xe2x80x94OH, xe2x80x94COOH, a halogen, xe2x80x94NH2, xe2x80x94OH.
For example, the, at least one, nitroaromatic compound may be a compound selected from nitrotoluene, dinitrotoluene, trinitrotoluene and their derivatives or a mixture of these compounds.
According to the invention, the method of the present invention may also comprise, before the contact step, a step consisting of neutralising the pH of the nitroaromatic compound solution or the soil such that the contact with the biomass, for example a Penicillium strain, can be performed at a pH of 4.5 to 6.5.
According to the invention, the contact may for example be performed in the presence of glucose at a concentration of 0.5 to 50 g of glucose/l of said solution.
According to the invention, the Penicillium strain may be a common Penicillium strain. For example, the Penicillium sp. LCM strain registered under the number I-2081 at Collection Nationale de Cultures de Microorganismes (CNCM) kept by Institut Pasteur in France may be used in the method according to the present invention. The invention also relates to the Penicillium sp. LCM strain registered under number I-2081 at the CNCM kept by Institut Pasteur in France.
According to the invention, the biomass of the Penicillium strain may be obtained using conventional microbiological methods to form a biomass, for example using Penicillium culture in a culture medium that is preferentially rich, solid or liquid, preferentially liquid, at a suitable temperature and pH to enable an optimal metabolism for the Penicillium strain.
A culture medium that can be used to develop the biomass is given in the examples below.
When a sufficient quantity of biomass is obtained, and when the culture medium is liquid, the biomass may for example be recovered by filtration or by centrifugation, advantageously by filtration.
Preferentially, in the method according to the present invention, the biomass is used fresh, which does not exclude any other use.
The biomass placed in contact with the solution or soil must be sufficient in quantity to enable the mineralisation of said nitroaromatic compound. The quantity of biomass may be determined for example as a function of the quantity of nitroaromatic compounds to be mineralised or the required mineralisation rate. This quantity of biomass may for example be determined using samples of the nitroaromatic compound solution mixed with variable quantities of biomass, at a suitable temperature and pH to enable a preferentially optimal metabolism of the Penicillium strain.
According to the invention, the contact may be carried out either by mixing the biomass and the nitroaromatic compound solution or by passing said solution through the biomass, said biomass being retained in, or on, a substrate such that said solution is able to pass through, to enable the mineralisation of the nitroaromatic compound it contains.
When the contact is carried out by mixing the biomass with the nitroaromatic compound solution, this contact may be carried out for example in a conventional reactor such as a fermenter, particularly comprising for example a system to stir or mix the solution and the biomass and a heating system. This type of contact is useful for example for a semi-continuous system, or batch system, for treating a nitroaromatic compound solution using the method according to the present invention.
When the contact is carried out by passing the nitroaromatic compound solution through the biomass, the biomass may be retained in, or on, a substrate. Indeed, advantageously, Penicillium in culture forms aggregates or pellets, making it possible to separate it easily from a solution, for example, by filtration.
According to the invention, the substrate may be for example a column or a conventional reactor enabling continuous passage of the nitroaromatic compound solution through the biomass, without carrying said biomass. The biomass may be retained for example by means of a grid, a fabric, or a mesh of organic or inorganic fibres which allows the passage of the solution, but not the biomass. This column or reactor may also comprise a system to stir the solution and the biomass and a heating system. This type of contact is useful for example for a system for the continuous treatment of a nitroaromatic compound solution using the method according to the present invention.
When the solution to be treated is a surface water, this contact may be carried out by simply dispersing the biomass and the Penicillium strain in the surface water.
According to the invention, when placed in contact with a nitroaromatic compound solution, the biomass may be mixed with the solution of said compound by means of a conventional stirrer, for example a rotary stirrer, and at a suitable stirring speed, for example from 50 to 250 rpm. The stirring may be maintained throughout the contact time between the Penicillium biomass and the nitroaromatic compound solution so as to optimise the contact.
According to the invention, the contact step is carried out under suitable conditions for a mineralisation of the nitroaromatic compound(s) by the Penicillium strain. Conventional preliminary Penicillium growth tests on samples of the nitroaromatic compound solution at different temperatures, the other parameters, for example the pH, being constant make it possible to determine this permissive temperature. It is important to note that Penicillium can develop even at a temperature of approximately 4xc2x0 C. In this way, the method according to the present invention particularly offers the advantage of the possibility to use it in a wide temperature range.
This permissive temperature may be for example approximately 10 to 35xc2x0 C., for example 15 to 35xc2x0 C. The inventors noted satisfactory mineralisation at a temperature from 20 to 30xc2x0 C., in particular at a temperature from 25 to 28xc2x0 C. Advantageously, the temperature may be maintained practically constant during the contact time so as to optimise the mineralisation.
According to the invention, the contact step is preferentially carried out at a permissive pH for the Penicillium strain metabolism. Tests such as those mentioned above but at a constant temperature and at different pH values may be carried out to determine this permissive pH. Generally, this pH may be from 5 to 7.5, for example from 5 to 6 and for example approximately 5.5.
Naturally, the pH of the solution varies during the process according to the invention since the nitro groups of the nitroaromatic compound may be reduced during the mineralisation of said compound. This variation in the pH does not affect the method according to the present invention.
However, according to a variant of the present invention, the pH of the solution may be kept practically constant during the contact step, at the above-mentioned values, by means of a conventional buffer such as a phosphate or citrate buffer. This buffer may be added to the soil or to the solution before or during the contact step of the method according to the present invention.
According to the invention, the contact may be carried out in the presence of a source of carbon for the Penicillium strain. This source of carbon is a general Penicillium metabolism inductor, and it favours the mineralisation of the nitroaromatic compound by this micro-organism. This source of carbon may be added in sufficient quantity to optimise the Penicillium metabolism, particularly the mineralisation of the nitroaromatic compound. This source of carbon may be for example selected from the group comprising glucose, glucose polymers, molasses, corn hydrolysates, etc. The glucose polymers comprise for example starch. The source of carbon may be added for example to the solution to be treated in the reactor, or, in the case of surface water, or a soil, directly into said water or onto said soil.
In a continuous solution treatment system, the reactor may also comprise a system to supply the source of carbon to the reactor.
According to the invention, when the source of carbon is glucose, and the nitroaromatic compound solution is a saturated solution of said compound for example a saturated solution of TNT, DNT, a derivative of said compounds or a mixture of said compounds or their derivatives, the glucose concentration during contact, in the solution, may be advantageously from 2 to 25 g/l, for example 15 g/l.
An optimal concentration of the source of carbon may be determined for example by tests of the method according to the invention, on samples of the nitroaromatic compound solution such as those mentioned above.
According to the invention, the suitable conditions mentioned above may also comprise the addition of Penicillium regulating or inducing additives in the solution or on the soil to be treated, such as magnesium sulphate, sodium nitrate, potassium chloride, iron sulphates, etc., trace elements, and as a general rule any additive known to favour the Penicillium metabolism.
Advantageously, according to the method according to the invention, the contact is carried out such that there is optimum contact between the Penicillium and the solution and will preferentially comprise stirring when carried out in a reactor.
Excess Penicillium biomass may induce the formation of an aggregate, or xe2x80x9ccakexe2x80x9d, which may have an adverse effect on the optimum contact between the micro-organism and the solution.
Advantageously, according to the present invention, the Penicillium biomass is placed in contact with the nitroaromatic compound solution at a concentration of 50 to 800 g in fresh biomass weight per litre of solution of said compound, for example approximately 150 g/l.
When the method according to the present invention relates to a method for eliminating a nitroaromatic compound present in a soil, the suitable conditions for a mineralisation of the, at least one, nitroaromatic compound present in said soil by the Penicillium strain are, of course, the favourable conditions for the Penicillium strain metabolism to mineralise the nitroaromatic strain to be eliminated. These conditions are particularly those described above.
It is also important to note that the Penicillium strain grows better in a wet medium.
Also, advantageously, this method according to the invention may comprise, before, during or after the step consisting of the contact of the soil to be treated with the biomass, a step consisting of the inundation of said soil with an aqueous solution, so as to form a nitroaromatic compound solution.
The method to mineralise a nitroaromatic compound in solution according to the invention and described above may then be applied.
Those skilled in the art will easily understand that the wetting of the soil may be natural, particularly by rainwater. In this case, the step consisting of the inundation of said soil with an aqueous solution will be carried out naturally with rainwater.
According to the invention, the contact of the soil, or the solution formed by inundating the soil, may be carried out by dispersing a Penicillium strain on the soil to be treated, advantageously a biomass of the Penicillium strain, before or after the inundation of said soil. Naturally, the soil may be inundated with an aqueous solution containing the Penicillium biomass.
A source of carbon, a pH buffer and an additive such as those described above may also be dispersed on the soil or during the contact with the Penicillium strain.
The method according to the invention is advantageous in relation to the methods of the prior art. Indeed, the inventors particularly demonstrated that the Penicillium strain made it possible to mineralise over 75% of an aromatic compound such as those described above, for example TNT, DNT and their derivative, easily, while the microbiological methods according to the prior art rarely exceed 10% mineralisation of said compounds. This mineralisation corresponds to a total or almost total degradation, or biodegradation of the nitroaromatic compound.
In addition, unexpectedly, the method according to the invention makes it possible to treat a solution with the above results even when it is saturated with nitroaromatic compound, i.e. it contains approximately 100 mg/l and up to 120 mg/l, of nitroaromatic compound, for example TNT, DNT and their derivatives. This concentration is very high and initially inhibited the use of a micro-organism to eliminate these compounds.
In addition, the method according to the invention is a non-polluting method since it generates little or no intermediate metabolites from the nitroaromatic compound(s), it does not require polluting chemicals and it uses a harmless micro-organism.
In addition, the method according to the invention is a very economical method, since it uses a micro-organism strain which is not expensive, commercially available and grows easily, even at temperatures of around 4xc2x0 C.
In addition, Penicillium tends to form pellets which can be extracted very easily from a solution, for example by simple filtration, which facilitates treatments of solutions, such as industrial effluents, for example in continuous mode.
Numerous other advantages and characteristics of the present invention can be seen by those skilled in the art upon reading the following illustrative and non-restrictive examples, with reference to the appended figures.